Human recombinant dpp 4

The enzyme, human recombinant DPP-IV (Lot: D4943-1VL), expressed in baculovirus-infected S/9 cells, the substrate, Gly-Pro p-nitroanilide (Gly-Pro pNA; Lot: G0513), and streptozotocin (STZ; Lot: V900890) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antidiabetic drug sitagliptin (Januvia 100 mg; Merk Sharp and Dohme Ltd., Hertfordshire EN11 9BU, Hertford Road, Hoddesdon, UK) was purchased at a drugstore in China. Other reagents, such as Folin–Ciocalteu (Lot: 73104861), gallic acid (Lot: XW01499171), and Coomassie Blue R-250 (Lot: 71011381) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lapsang souchon (LS), a typical Chinese black tea, was obtained from a tea market in Hangzhou.

DPP-4 inhibition was assessed, as previously described [42 (link),43 (link)], based on a continuous fluorometric assay method in which the substrate Gly-Pro-AMC (Gly-Pro-7-amido-4-methylcoumarin hydrobromide) was split by DPP-4 to release fluorescent aminomethyl coumarin (AMC). The liberated AMC was continuously monitored using an excitation wavelength of 360 nm and an emission wavelength of 460 nm using an EnVision microplate reader (PerkinElmer, Waltham, MA, USA). The reaction mixture consisted of different concentrations of the synthesized hybrids 10a–j, 20 µU/µL of recombinant human DPP-4 (Sigma Aldrich, St. Louis, MO, USA), 10 µM Gly-Pro-AMC (Sigma Aldrich, St. Louis, MO, USA) and assay buffer (150 mM NaCl, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.12 mg/ml bovine serum albumin (BSA)) and pH of 7.5. The assay was carried out in quadruplicate. IC₅₀ was calculated using GraphPad Prism 8 software (San Diego, CA, USA). Similar to the DPP-4 assay, DPP-8 and DPP-9 inhibition assay was performed and the pH of the assay buffer was 8.